Help with ChIP analysis using Taqman - (Jan/10/2007 )
My lab has recently started doing ChIP experiments, using Taqman real-time PCR to analyse our data. However, I’m a little bit confused as to exactly how ChIP data should be analysed, and what controls are necessary to show that any enrichment observed is genuine.
Prior to carrying out immunoprecipitations (IPs) I remove a chromatin sample which I call ‘Input’. The chromatin sample is then divided into two, half of which I IP with an antibody against my protein of interest (+Ab), whilst no antibody is added to the other half (No Ab). Both samples are then pulled down with beads and cleaned up as for standard ChIP protocol.
I have a Taqman probe for my region of interest (which I suspect binds the transcription factor I’m interested in), as well as a positive control probe (for a region I know binds the TF) and a negative control probe (which doesn’t bind the TF).
Using my taqman data, would it be best to calculate (+Ab / Input) or (+Ab / No Ab) for each probe? After I get these values, would it be okay for me to normalize everything to my negative control probe? (i.e. Showing enrichment compared to a region that doesn’t bind the TF).
Also, what are the pros and cons for using either the standard curve method or delta Ct method for Taqman. I’ve currently been using the standard curve method, but it gets quite time consuming (and costly!) constructing a standard curve every time. It seems that the delta Ct method would involve a lot less work, but is it as reliable?
Any help would be very much appreciated!
For the second part of your question, you could use ddCT if your pcr rxn is efficient. This is based upon the slope of your standard curve.
For the first part, I believe you shoudl analyze your data IP/input vs. Control AB IP/input.